When wounded, eukaryotic cells reseal their plasma membrane in a few seconds. This process is essential to avoid loss of cytosolic factors, and for restoring the critical barrier between the intracellular and extracellular environments. Calcium influx through wounds triggers lysosomal exocytosis, an event required for plasma membrane repair. Exocytosis was thought to mediate plasma membrane resealing by addition of an endomembrane patch, or by relieving membrane tension, facilitating spontaneous bilayer restoration. However, it recently became clear that calcium influx also triggers the repair of lesions caused by pore-forming proteins. When inserted in the plasma membrane, pore-forming proteins generate stable lesions that cannot be resealed by a patch, or by reducing membrane tension. An investigation of this process revealed that calcium influx in injured cells markedly stimulates endocytosis, with a kinetics that coincides with cell resealing. Additional results suggested that trans-membrane pores and mechanical lesions are removed from the plasma membrane by endocytosis, and that this process requires exocytosis of a lysosomal enzyme, acid sphingomyelinase. These new findings represent a major conceptual advance in our understanding of plasma membrane repair, since they indicate that the role of lysosomal exocytosis is to release a critical hydrolase that acts on the cell surface, and not to add a patch or relieve membrane tension. We hypothesize that calcium entry triggers exocytosis of lysosomal acid sphingomyelinase, which cleaves sphingomyelin at the cell surface, generating ceramide and inducing formation of endosomes that carry the lesions into the cells for degradation. To test this hypothesis, we will pursue two specific aims: 1) Determine if exocytosis of lysosomal acid sphingomyelinase during plasma membrane wounding leads to ceramide generation and endosome formation, and whether this injury repair pathway is present in muscle fibers;2) Determine the intracellular fate of the endosomes generated during plasma membrane wounding with pore-forming toxins. In addition to clarifying how cells survive attack by membrane damaging agents produced by pathogens, this project will provide new insight on mechanisms underlying the pathology of serious human diseases, including lysosomal storage diseases and muscular dystrophy. PUBLIC HEALTH RELEVANCE: Plasma membrane repair is essential for cellular survival after injury. The goal of this proposal is to investigate the mechanism by which mammalian cells remove wounds on their membrane by endocytosis. The results will clarify how cells survive attack by toxins produced by pathogens, and will increase our knowledge of defects leading to serious human diseases including lysosomal storage diseases and muscular dystrophy.